Buthionine sulfoximine and a metallodrug for the treatment of cancer, HIV or a rheumatic disease

ABSTRACT

There is disclosed buthionine sulfoximine (BSO) for use in a method of treatment of cancer, HIV or a rheumatic disease. The method comprises administering (a) BSO at a dosage of less than or equal to 50 mg/kg, and (b) a metallodrug at a clinically-acceptable dosage.

RELATED APPLICATIONS

This application is a § 371 National Phase Application of InternationalApplication No. PCT/EP2018/057557, filed on Mar. 23, 2018, nowInternational Publication No. WO 2018/172559, published on Sep. 27,2018, which International Application claims the benefit under 35 USC119(e) of U.S. Provisional Application No. 62/475,654, filed on Mar. 23,2017, both of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention provides buthionine sulfoximine (BSO) for use as amedicament with a metallodrug, methods of treatment using BSO and ametallodrug, kits comprising BSO and a metallodrug, and compositionscomprising BSO and a metallodrug.

BACKGROUND

Reduction of immune activation represents a promising frontier fordevelopment of novel therapeutics for cancer and HIV/AIDS. Thesediseases are characterized by malignant immune hyperactivation linked toimmune exhaustion. Especially in the context of HIV/AIDS, however,therapeutic interventions aimed at blocking immune hyperactivation haveproduced mixed or limited results in clinical settings (Shytaj andSavarino, 2015).

A new emerging wave of research in anticancer therapy exploits oxidativestress and nitrosative stress produced by metallodrugs, such asgold(I)-containing metallodrugs and platinum(II)-containingmetallodrugs. Other groups of metallodrugs which are thought to beuseful are gold(III)-containing metallodrugs and platinum(IV)-containingmetallodrugs. Indeed, cisplatin, a platinum(II)-containing metallodrug,is currently used as a chemotherapeutic agent. In addition, auranofin, agold-containing metallodrug, has been shown to be useful in thetreatment of rheumatoid arthritis, and has been considered in anticancerstrategies.

A further area of research has been the combination of metallodrugs withother pharmaceuticals to potentiate oxidative stress. There are two maincellular anti-oxidative pathways, i.e. the thioredoxin (Trx)/thioredoxinreductase (TrxR) system and glutathione (GSH). These machineries are inpart overlapping and are capable of backing each up other (Benhar et al.JCI 2016). In case one of the two systems is blocked, the other maybecome up-regulated, thus providing at least partial compensation forthe intracellular antioxidant machinery. Some metallodrugs, andparticularly gold-containing compounds, such as auranofin, are known toinhibit TrxRs. In addition, metallodrugs have been shown to selectivelykill cells derived from multiple types of cancers when administered inthe presence of buthionine sulfoximine (BSO). BSO inhibitsgamma-glutamyl cysteine synthetase, a limiting enzyme in the glutathionesynthetic pathway (Griffith et al. J Biol Chem. 1979), and thereforeinhibits the other of the two anti-oxidative pathways. Thus, thepossibility of combining a metallodrug, such as auranofin and cisplatin,with BSO for treating diseases might be useful through a) the ability toinduce oxidative and nitrosative stress, and b) the ability tocounteract the cellular oxidative machinery which activates in order tolimit the effects of oxidative stress.

Malaker et al. studied the effect of BSO, including submicromolarconcentrations, on glutathione levels following glutathione depletion bydiethylmaleate. This study was conducted with a view to use BSO inanticancer treatment with etanidazole, in order to radiosensitize cells(Malaker et al. Int J Radiat Oncol Biol Phys. 1994). In order to achievea 90% decrease in glutathione levels, 6 μM BSO was required, and theauthors also reported a possible problem with under-dosing of BSO.

BSO has been used in the treatment of cancer, particularlyneuroblastoma, in combination with melphalan. For example, Villablancaet al. (2016) administered a 3 g/m² bolus, followed by 24 g/m²/day ofBSO with escalating doses of melphalan (20-125 mg/m²) to patients withrecurrent or resistant high-risk neuroblastoma.

Anderson et al. (2015) administered BSO as a fixed loading dose of 3g/m² followed by a 72-hour continuous infusion of 0.75 g/m² or 1 g/m²,with 15 mg/m2 of melphalan, in order to determine the effect forchildren with recurrent or refractory high-risk neuroblastoma.

O'Dwyer et al. (1992) administered 1.5 g/m² BSO at 12 hour intervals forsix doses, alone or with 15 mg/m² melphalan after the fifth dose. Markeddepletion of cellular GSH was noted.

From in vitro experiments, the combination of gold(I)-containingcompounds, such as auranofin, and BSO is known to limit immuneactivation (Vint et al., 1993), but the effects of this drug combinationon immune activation in the in vivo cancer setting have, however,remained unexplored. Using a mathematical model it has been possible topredict that limitation of immune activation might lead to a “bottleneckeffect” which can favor the maintenance of immune responses that aremore “focused” towards the antigens less likely to be mutated. Theseantigens are statistically more represented in a typical viralpopulation due to their being more evolutionary conserved and, thus, thelymphocytic clones targeting them are more likely to be overrepresentedand survive the aforementioned “bottleneck effect” (Benhar et al. JCI2016). Thus, it appears that induction of an efficient immune responsethrough lymphorestriction could have played a pivotal role in thetherapeutic effects of auranofin and BSO in the in vivo studiesconducted so far. The availability of manageable agents able tocounteract malignant immune hyperactivation in cancer represents a newresearch priority.

BSO in combination with platinum-containing metallodrugs, such ascisplatin, for the treatment of cancer, has also been explored.Cisplatin is a chemotherapeutic agent, used for treating, for example,malignant mesothelioma, bladder, ovarian, testicular, non-small celllung, endometrial, penile, head and neck, anal, biliary tract,hepatocellular and gastric cancer, non-Hodgkin's lymphoma, malignantmelanoma and neuroblastoma, amongst others. Dosages up to 100 mg/m² areconsidered safe (Prescriber's Digital Reference—cisplatin;http://www.pdr.net/drug-summary/Cisplatin-cisplatin-1472).

Tsutsui et al. (1986) administered 5 mmol/kg (equivalent to 111.5 mg/kg)BSO 12 hours before administration of cyclophosphamide, cisplatin orbleomycin. Cisplatin was administered at an amount of 2.5, 5, 7.5 or 10mg/kg. It was found that BSO increased the anti-cancer effects of thesethree chemotherapeutic agents.

Saikawa et al. (1993) aimed to evaluate the enhancement of the antitumoractivity of cisplatin by BSO in vitro and in vivo. In vitro,pre-treatment of MKN-28 and MKN-45 cells with BSO (5, 10 and 25 mM)increased the antitumor activity of cisplatin (0.1, 0.3, 1.0 and 3.0μg/ml for MKN-28 and 0.01, 0.1, 0.3 and 1 μg/ml for MKN-45). In vivo,antitumor effects of cisplatin (6 or 9 mg/kg) were enhanced bypre-treatment with BSO, which was administered intraperitoneally at adose of 500 mg/kg.

Similarly to neoplastic conditions, infections by lentiviruses (a viralgenus comprising human immunodeficiency virus (HIV)) appear to besusceptible to the auranofin/BSO combination, and the administration ofauranofin (0.4 mg/kg/day) and BSO (450 mg/kg) to macaques infected withthe HIV homolog SIVmac251 in combination with antiretroviral therapy(ART) induced a functional cure of the disease following suspension ofall therapies (Shytaj et al. Retrovirology 2013; Shytaj et al. J Virol.2015).

This functional cure consisted of prolonged control of viral load for upto two years: the macaque that was not subjected to euthanasia hasremained healthy and in good condition four and a half years aftersuspension of therapy. This functional cure was associated with higherHIV-specific responses than those observed in auranofin/BSO-untreatedmacaques in which ART had been suspended. In the case of HIV, not onlycan limitation of immune activation result in increased immuneresponses, but also in a decrease of the main HIV reservoir, i.e.latently infected memory CD4⁺ T-cells harboring silent copies of theviral DNA which cannot be attacked by drugs or recognized by the immunesystem. Similarly to cancer cells, these cells proliferate within theorganism despite the high-level suppression of viral replicationprovided by ART (Benhar et al. JCI 2016). There is a correlation betweenthe magnitude of the viral reservoir and the level of immune activationin HIV-infected individuals (Shytaj and Savarino Retrovirology 2015).Immune activation per se represents a problem in ART-treated HIV⁺individuals, as high levels thereof are associated with a higherincidence of non-AIDS defining events, such as cardiovascular diseaseand cancer. Strategies aimed at limiting immune activation during ARTare a so far unmet medical need.

Auranofin has been used for decades in treatment of rheumatoidarthritis, a condition in which it showed some degree of efficacy,according to meta-analysis studies (Suarez-Almazor et al. CochraneDatabase Syst Rev. 2000). However, one limitation of its use resides inthe fact that, for safety, the drug cannot be administered for periodslonger than nine months. Moreover, doses greater than 9 mg/day areconsidered unsafe, as they can cause gold toxicity (Prescriber's DigitalReference—Auranofin;http://www.pdr.net/drug-summary/Ridaura-auranofin-795). It is,therefore, highly desirable to increase its therapeutic potency duringthe treatment period, and BSO is considered to be an additive option toauranofin. However, the use of BSO is limited by the costs andcomplexity of its synthesis (an explosive step is involved in thesynthetic pathway; Griffith et al. J Biol Chem. 1979) and by theextremely high dosages adopted so far in vivo (Bailey et al. J ClinOncol. 1994, O'Dwyer et al. J Clin Oncol. 1996, Bailey et al. J NatlCancer Inst. 1997; Anderson et al. Pediatr Blood Cancer. 2015). Whilethese dosages result in peak plasma drug concentrations matching thoseat which the best in vitro effects have been documented (Meister.Pharmacol Ther. 1991; Vint et al. Cell. Immunology 1993; Shytaj et al.J. Virol 2015), they increase the risk of side effect and the problemswith producing enough BSO for clinical use, as shown by the very longhiatus between clinical trials of this drug.

BSO has previously been reported to decrease glutathione whenadministered in the low micromolar concentration range; however, moreprofound glutathione depletion has been observed at higherconcentrations, a phenomenon which has prompted investigators to testthe drug in vivo at dosages resulting in massive glutathione depletion(by approx. 80%), in order to maximize the chances for success, asdiscussed above. As also discussed above, high BSO concentrations (up tothe millimolar range) have also been largely adopted in in vitroexperiments aimed at chemosensitising cells to antineoplastic andanti-HIV latency agents. Accordingly, high dosages (namely, inducingpeak plasma concentrations of 250 μM) of BSO have been previouslyspecified.

Wang et al. (Oncotgarget 2017) explored the potential anticancerapplication of a combination of low dose/concentration BSO (producing apeak plasma concentration of 1 μM) and auranofin. However, the decreasein the concentration of BSO to the low micromolar range was compensatedby an increase of auranofin to concentrations which were far superior6-fold) to those acceptable in a clinical setting, and at whichauranofin has off-target effects, including BSO-like induction of adecrease in glutathione levels (You et al. Molecular Medicine Reports2015). Moreover, in the highly resistant murine mammary carcinoma cellline that they adopted, Wang et al. were not able to detect any directlycytotoxic/anticancer effect of the auranofin/submicromolar BSOcombination, and analysed the aforementioned drug combination in termsof radiosensitization of tumor cells. The chemosensensitisation of thecells, by the drug combination, to the cytotoxic effects of ametallodrug at clinically-acceptable concentrations was not analysed.

So far, therefore, it is considered that high therapeutic dosages of BSOare required for treatment with metallodrugs, or that high doses ofmetallodrugs are required to compensate for any low dosages of BSO.Indeed, it has been considered that low dosages of BSO have an oppositeeffect to metallodrugs. For example, Mayer et al. showed that BSO couldreduce the nephrotoxicity of the metallodrug cisplatin in rats (Mayer etal. J. Cancer Res. Clin. Oncol. 1989). Therefore, the interest in ananticancer combination of a metallodrug and BSO has decayed throughoutthe years despite promising results in vitro and in vivo.

Thus, there is a need to provide a treatment which overcomes theproblems and disadvantages discussed above. In particular, it isdesirable to provide a treatment which overcomes the problem ofoff-target effects, malignant immune hyperactivation and inefficientimmune responses. It is also desirable to overcome the problem of thecost and complexity of production of BSO, and inconvenient treatmentregimes.

SUMMARY OF THE INVENTION

The present invention provides solutions to the problems discussed abovebecause it has now, surprisingly, been found that dosages of BSO10-1000-fold lower than previously used, together withclinically-acceptable amounts of metallodrugs, are useful for killingcancer cells and sensitising cancer cells to chemotherapeutic agents,for limiting malignant immune hyperactivation and for improving theefficiency of immune responses. Thus, the ratio of BSO to metallodrugcan be decreased, and the possibility of off-target effects in patientscan be limited.

The decreased dosages of BSO provide for the treatment of diseasesthrough an anti-inflammatory effect involving the reduction of immunehyperactivation, or through a direct cytotoxic effect. For example, thereduced dosages of BSO of the present invention have the potential tolimit the proliferation of HIV reservoir cells. The reduced dosages ofBSO also provide a direct effect on targets of the immune system, inparticular, killing and chemosensitising of cancer cells. BSO can beadministered separately, sequentially or simultaneously with themetallodrug. Thus, the cost of treatments using BSO can be reduced, andthe complexity of the production of BSO can be mitigated. Moreover, thereduction in the amount of BSO means that BSO and a metallodrug could beprovided in a single composition, thereby reducing the inconvenience oftreatment to the patient and providing further, more convenient, routesof administration.

The present invention also relates to methods of treating patients withBSO and a metallodrug, to kits comprising BSO and a metallodrug, and tocompositions comprising BSO and a metallodrug. The methods, kits andcompositions, provide for BSO to be administered at a dosage asdescribed above, namely, 10-1000-fold lower than previously useddosages.

Thus, the invention provides BSO for use in a method of treatment,separately, simultaneously or sequentially with a clinically-acceptableamount of a metallodrug, wherein the method comprises administering BSOat an amount 10-1000-fold lower than previously used.

The invention also provides BSO for use in a method of treatment ofcancer, HIV or a rheumatic disease, wherein the method comprisesadministering (a) BSO at a dosage of less than or equal to 50 mg/kg, and(b) a metallodrug at a clinically-acceptable dosage.

The treatment may be of cancer, and the BSO may be administered at adosage of less than or equal to 9 mg/kg.

In particular, the cancer may be haematological malignancy, Hodgkin'slymphoma, non-Hodgkin's lymphoma, lung carcinoma, prostate cancer,hepatocellular carcinoma, breast cancer, glioblastoma, uterine cervixcarcinoma or Kaposi's sarcoma.

The BSO may be administered at a dosage of between 0.45 mg/kg and 9mg/kg. Alternatively, the treatment may be of HIV or a rheumaticdisease.

In particular, the treatment may be of rheumatic arthritis or lupus.

The BSO may be administered at a dosage of between 0.45 mg/kg and 45mg/kg.

The metallodrug may comprise a gold-containing metallodrug and/or aplatinum-containing metallodrug.

The treatment may be for rheumatic diseases or HIV, and the metallodrugcomprises a gold-containing metallodrug.

The gold-containing metallodrug may comprise auranofin, aurothioglucose,sodium aurothiopropanolsulfonate and/or aurothiomalate, and/or theplatinum-containing metallodrug comprises cisplatin, carboplatin,oxaliplatin, nedaplatin, lobaplatin, heptaplatin and/or satraplatin.

The gold-containing metallodrug may comprise auranofin, aurothioglucose,sodium aurothiopropanolsulfonate and/or aurothiomalate, and preferablymay be auranofin.

The ratio of BSO to gold-containing metallodrug may be 1:0.25 to 1:10,and/or the ratio of BSO to platinum-containing metallodrug may be 1:5 to1:25.

Preferably, the treatment is of cancer and the ratio of BSO togold-containing metallodrug is 1:1 to 1:10 and/or the ratio of BSO toplatinum-containing metallodrug is 1:5 to 1:25.

Alternatively, the treatment may be of HIV or a rheumatic disease, andthe ratio of BSO to gold-containing metallodrug may be 1:0.25 to 1:1.

Preferably, the metallodrug comprises auranofin administered at a dosageof 3-9 mg/day.

Alternatively, the treatment is of cancer and the metallodrug comprisescisplatin administered at a dosage of less than or equal to 100 mg/m².

The invention also provides a method of treating cancer, HIV or arheumatic disease, wherein the method comprises administering to apatient in need thereof (a) BSO at a dosage of less than or equal to 50mg/kg, and (b) a metallodrug at a clinically-acceptable dosage.

The invention further provides a method of treatment of cancer, HIV or arheumatic disease, wherein the method comprises administering to apatient in need thereof (a) BSO at a dosage sufficient to achieve a peakplasma concentration of 0.5-20 μM, and (b) a metallodrug at aclinically-acceptable dosage.

Preferably, the BSO is administered at a dosage sufficient to achieve amean plasma concentration of 0.05-2 μM.

The method may be for the treatment of HIV or a rheumatic disease.

Alternatively, the method is for the treatment of cancer and the BSO isadministered at a dosage sufficient to achieve a mean plasmaconcentration of 0.05-0.8 μM.

The invention also provides a kit comprising BSO and a metallodrug,wherein (a) the BSO is at a dosage of less than or equal to 5000 mg, and(b) the metallodrug is at a clinically-acceptable dosage.

The invention further provides a kit comprising BSO and a metallodrug,wherein the metallodrug is at a clinically-acceptable dosage, and theratio of the BSO to the metallodrug in the kit is between 1:0.25 and1:25.

The invention also provides a pharmaceutical composition comprising BSOand a metallodrug, wherein the metallodrug is present at aclinically-acceptable amount, and the BSO is present at a dosage of lessthan or equal to 50 mg/kg, and wherein the pharmaceutical compositioncomprises a pharmaceutically-acceptable carrier, diluent and/orexcipient.

The invention further provides a pharmaceutical composition comprisingBSO and a metallodrug, wherein the metallodrug is present at aclinically-acceptable amount and the ratio of BSO to the metallodrug isbetween 1:0.25 and 1:25, and wherein the pharmaceutical compositioncomprises a pharmaceutically-acceptable carrier, diluent and/orexcipient.

“Clinically-acceptable amount”, as used herein, means an amount which isrecognised as being safe by the relevant medical authorities. Guidanceon clinically-acceptable dosages can be found, for example, at thePrescriber's Digital Reference (http://www.pdr.net/).

A “metallodrug”, as used herein, is a pharmaceutical which contains ametal as an active ingredient, for example, silver, zinc, platinum orgold. They are commonly used as anti-cancer and antimicrobial drugs.Metallodrugs are well-known in the art and include gold-containing,platinum-containing, silver-containing and zinc-containing drugs.Specific metallodrugs include cisplatin, auranofin, carboplatin andsilver sulfadiazine.

“Mean plasma concentration”, as used herein means the average plasmaconcentration, calculated as Area Under the Curve of aconcentration-time curve after a single dose or in steady state.

“Peak plasma concentration”, as used herein, means the highest plasmaconcentration of a drug achieved after administration of the drug.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the viability of activated primary CD4⁺T-cells treated with different concentrations of auranofin andbuthionine sulfoximine (BSO), measured with the MTT assay. The cellswere pre-incubated with the drugs for 24 hours and then activated withphytohemagglutinin (PHA) and assayed for viability 72 hours postactivation (A) or the cells were activated with αCD3-αCD28 beads andthen treated for 48 h with different combinations of auranofin and BSO(B). In (A), the effects of chloroquine are shown as a matter ofcomparison with a drug adopted in the clinic to limit immunehyperactivation. All concentrations are expressed in μM.

FIG. 2 is a graph showing the viability of Supt-1 T-cells treated withdifferent concentrations of auranofin (AU) or buthionine sulfoximine(BSO). Viability was measured 72 hours after treatment with the drugs atthe indicated concentrations with the MTS assay. Absorbance values werenormalized over untreated controls and expressed as percentage.BSO=buthionine sulfoximine. Concentrations are expressed in μM. Data areexpressed as mean±SEM and were analysed by the Turkey posttest followingOne Way ANOVA. *P<0.05; **P<0.01 ***P<0.001; ****P<0.0001. NS:non-significant. The synergistic index (SI) is expressed wheresignificant differences exist in comparison with the single drugtreatments.

FIG. 3 is microphotographs of Supt-1 T-cells treated with differentconcentrations of auranofin or buthionine sulfoximine.

FIG. 4 is a graph showing the viability of Jurkat T-cells treated withdifferent concentrations of cisplatin, auranofin and/or buthioninesulfoximine (BSO). Viability was measured 48 hours after treatment withthe drugs at the indicated concentrations with the MTT assay. Absorbancevalues were normalized over untreated controls and expressed aspercentage. Concentrations are expressed in μM. Data are expressed asmean±SEM and were. Asterisks show the significant differences [*q<0.05;**q<0.01; q is the P value corrected for multiple comparisons bycontrolling the False Discovery Rate (threshold forsignificance=q=0.05)]. NS: non-significant. The synergistic index (SI)is expressed where significant differences exist in comparison with thesingle drug treatments.

FIG. 5 is microphotographs of Jurkat T-cells treated with differentconcentrations of cisplatin, auranofin or buthionine sulfoximine (BSO).

FIG. 6 is a graph showing the viability of Supt-1 T-cells treated withdifferent concentrations of cisplatin or buthionine sulfoximine (BSO).Viability was measured 72 hours after treatment with the drugs at theindicated concentrations with the MTS assay. Absorbance values werenormalized over untreated controls and expressed as percentage.BSO=buthionine sulfoximine. Concentrations are expressed in μM;Asterisks show the significant differences [*q<0.05; **q<0.01; q is theP value corrected for multiple comparisons by controlling the FalseDiscovery Rate (threshold for significance=q=0.05)]. NS:non-significant. The synergistic index (SI) is expressed wheresignificant differences exist in comparison with the single drugtreatments.

FIG. 7 shows the results of numerical simulations of the Rong andPerelson model with programmed expansion and contraction of the viralreservoir. A simulation of the viral load (RNA copies/nil) and viralreservoir (L cells) dynamics in a human model is provided. The trend ofproductively infected cells (T*) is also shown. The simulation is basedon the five differential equations model in Rong and Perelson (2009).The peaks in the viral load correspond to the periods of activation oflatently infected CD4+ T-cells. For starting data see Rong and Perelson(2009). Different proliferation rates (p) are shown (day⁻¹),corresponding to different drug treatments, or absence of any treatment.The bottom three panels depict a scenario without the drugs, with p=1.4day⁻¹. The effects of BSO in combination with auranofin at plasma molarratios (BSO/auranofin) of 1:0.25 (top three panels), 1:0.5 (second lineof panels from top), 1:1 (third line of panels from top) are simulated.The plasma concentration of auranofin is 500 microM. The activationfunction adopted to simulate lymphocyte encounter with antigens isillustrated in Rong and Perelson (2009).

DETAILED DESCRIPTION

The present invention is based on the discovery that it is possible toachieve the same, if not improved, therapeutic effects in the treatmentof patients by using concentrations of BSO that are much lower thanpreviously used, together (separately, simultaneously or sequentially)with a clinically-acceptable amount of a metallodrug. The inventionrelates, in general terms, to the use of concentrations of BSO which aremuch lower than previously used, and a metallodrug, for treatingpatients. In particular, the dosages of BSO of the present invention are10-1000-fold lower than previously used, and this allows the ratio ofBSO to metallodrug to be increased.

Indeed, Example 1 shows that reduced concentrations of BSO, with ametallodrug at a clinically-acceptable dosage, have a limiting effect onimmune activation and have an anti-proliferative effect. As discussed inExample 1, the reduction of the peak plasma concentration of BSO to 2μM, with a clinically-acceptable dosage of a metallodrug, limited cellviability to a similar degree to previously used peak plasma BSOconcentrations of 250 μM. Submicromolar concentrations of BSO were alsoshown to have a direct anti-cancer effect (FIGS. 2 and 3), and asynergism (indicated by a synergistic index of less than 0.9) wasmeasured only for BSO concentrations in the submicromolar range. The BSOconcentrations of the invention were also shown to induce morphologicalchanges which are linked to poor cell viability (FIG. 3). Similareffects were seen using cisplatin instead of auranofin, at aclinically-acceptable dosage (FIGS. 4 and 6), and the anticancer effectwas similar to that produced by a concentration of BSO three orders ofmagnitude higher. Furthermore, it was found that BSO at concentrations10-1000-fold lower than previously used produced an increasedantineoplastic effect when used with both auranofin and cisplatin (FIGS.4 and 5).

The results of Examples 2 and 3 also show that concentrations of BSO inthe low micromolar or submicromolar concentration range, and incombination with auranofin, may well have an impact on the viralreservoir (for example, in HIV) which is at least similar to thatachieved by previously-used dosages.

BSO is administered to the patient at dosage 10-1000 times lower thanthe dosages currently used or postulated. In particular, BSO is given ata dosage of less than or equal to 45 mg/kg. In some embodiments, BSO isadministered at a dosage of less than or equal to 40 mg/kg, 35 mg/kg, 30mg/kg, 25 mg/kg, 20 mg/kg, 15 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.9 mg/kg,0.8 mg/kg, 0.7 mg/kg, 0.6 mg/kg, 0.5 mg/kg, 0.45 mg/kg, 0.4 mg/kg, 0.35g/kg, 0.3 mg/kg, 0.25 mg/kg, 0.2 mg/kg, 0.15 mg/kg or 0.1 mg/kg.

In some embodiments, BSO is administered at a dosage of at least 0.1mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4mg/kg, 0.45 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg 0.9 mg/kgor 1.0 mg/kg.

In preferred embodiments, BSO is administered at a dosage between 0.45mg/kg and 45 mg/kg. In other preferred embodiments, BSO is administeredat a dosage of between 0.45 mg/kg and 9 mg/kg. In some embodiments, BSOis administered at a dosage of between 0.45 mg/kg and 8 mg/kg.

The dosage of BSO can also be expressed in terms of the peak or meanplasma concentration to be achieved by the administration of BSO.Techniques for measuring the peak and mean plasma concentration are wellknown to the person skilled in the art. In particular, the peak and meanplasma concentrations of a drug are determined by measuring plasmalevels of the drug at set times after administration of the drug, forexample, every hour from time zero to 24 hours after administration.Plasma concentration-time profiles are plotted. Mean plasmaconcentration is determined by calculating the area under the profile(Area Under Curve; AUC) using the trapezoidal rule. Peak plasmaconcentration is the highest plasma concentration achieved after asingle dose of the drug. Plasma concentration of drugs can be measuredby well-known techniques, such as capillary electrophoresis/HPLC (Sandoret al., 1995) and inductively-coupled plasma-mass spectrometry (ICP-MS;Capparelli et al., 2017).

In some embodiments, BSO is administered at a dosage which achieves apeak plasma concentration of less than or equal to 25, 20, 15, 10, 5, 4,3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.25, 0.2, 0.15, 0.1 or 0.05μM. In some embodiments, the peak plasma concentration is greater thanor equal to 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 2, 3, 4, 5, 10, 15, and 20 μM. In some embodiments, the peakplasma concentration is less than 2 μM or less than 1 μM. In otherembodiments, the peak plasma concentration is 0.5-20 μM.

In some embodiments, BSO is administered at a dosage which achieves amean plasma concentration of less than or equal to 25, 20, 15, 10, 5, 4,3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.25, 0.2, 0.15, 0.1 or 0.05μM. In some embodiments, the mean plasma concentration is greater thanor equal to 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 2, 3, 4, 5, 10, 15, and 20 μM. In some embodiments, the meanplasma concentration is less than 2 μM or less than 1 μM. In otherembodiments, the peak plasma concentration is 0.05-2 μM. In furtherembodiments, the peak plasma concentration is 0.05-0.8 μM.

The metallodrug used in the present invention may be any metallodrug. Inparticular, metals, and particularly transition metals, are able toinduce oxidative stress through the formation of reactive oxygen species(ROS). They are also able to produce nitrosative stress through thegeneration of reactive nitrogen species (RNS). It is thought that thisoxidative and/or nitrosative stress can be potentiated by the inhibitionof cellular anti-oxidative pathways, such as the inhibition GSH by BSO,as glutathione is one of the main scavengers of ROS. Furthermore, someheavy metals being used and explored in pharmacotherapy (i.e. gold,platinum, silver, ruthenium and silver) are able to inhibit the Trx/TrxRanti-oxidative pathway, further potentiating the oxidative and/ornitrosative stress induced by metallodrugs. Thus, of the decreaseddosages of BSO of the invention (i.e. which provides as increased ratioof BSO to metallodrug) maintains the potentiation of the effect of ROSwhile reducing the amount of off-target side effects caused by BSO.

Interestingly, the ratios of BSO to auranofin or cisplatin (detailedfurther below) that can be achieved with the present invention overlap,such that it would be expected that BSO will exert synergistic effectsin combination with other metallodrugs, particularly those which arecapable of exerting pro-oxidant effects by similar mechanisms, and moreparticularly, other gold- or platinum-containing drugs, at similar molarratios.

In some embodiments, BSO is administered in combination with ametallodrug, such as a gold-containing, a silver-containing, azinc-containing and/or a platinum-containing drug. Examples ofgold-containing metallodrugs which may be used are auranofin,aurothioglucose, sodium aurothiopropanolsulfonate and aurothiomalate.Examples of platinum-containing metallodrugs which may be used arecisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, heptaplatinand/or satraplatin. In some embodiments, the metallodrug is auranofin orcisplatin.

In some embodiments, more than one metallodrug may be used. For example,more than one gold-containing metallodrug, more than oneplatinum-containing drug, more than one zinc-containing metallodrug, ormore than one silver-containing metallodrug may be used. In someembodiments, the more than one metallodrug may be a combination of oneor more gold-containing metallodrug, one or more platinum-containingdrug, one or more zinc-containing metallodrug and/or one or moresilver-containing metallodrug. Thus, in some embodiments, themetallodrug is one or more of auranofin, aurothioglucose, sodiumaurothiopropanolsulfonate, aurothiomalate, cisplatin, carboplatin,oxaliplatin, nedaplatin, lobaplatin, heptaplatin and/or satraplatin. Insome embodiments, a gold-containing metallodrug and aplatinum-containing metallodrug are used. Preferably, auranofin andcisplatin are used.

The metallodrug is administered at a clinically-acceptable amount. Inparticular, the use of metallodrugs for the treatment of diseases, andmaximum dosages thereof that are considered safe, are well known. Forexample, as mentioned above, gold-containing metallodrugs such asauranofin are considered unsafe at dosages greater than 9 mg/day(Prescriber's Digital Reference—Auranofin;http://www.pdr.net/drug-summary/Ridaura-auranofin-795). In embodimentswhere more than one metallodrug is used, the dosage of each metallodrugis a clinically-acceptable dosage.

Thus, for example, in embodiments comprising auranofin, the dosage isless than or equal to 9 mg/day. In other embodiments the dosage ofauranofin is 3-9 mg/day or 3-6 mg/day. The dosage of auranofin mayresult in a mean plasma concentration of less than or equal to 3 μM, 2μM or 1 μM. Preferably, the dosage of auranofin results in a mean plasmaconcentration of less than 1 μM. In some embodiments, the auranofin isadministered at a dosage sufficient to achieve a mean plasmaconcentration of 0.2-1 μM.

In embodiments comprising cisplatin, the clinically-acceptable dosage isless than or equal to 100 mg/m² (Prescriber's DigitalReference—cisplatin;http://www.pdr.net/drug-summary/Cisplatin-cisplatin-1472). In otherembodiments, the dosage of cisplatin is less than or equal to 75, 70,65, 60, 55, 50, 45, 40, 35, 30 or 25 mg/m². In some embodiments,cisplatin is administered at a dosage sufficient to achieve a meanplasma concentration of less than or equal to 50, 40, 30, 20, 10 or 5μM. In some embodiments, cisplatin is administered at a dosagesufficient to achieve a mean plasma concentration of 2-50 μM. In otherembodiments, cisplatin is administered at a dosage sufficient to achievea mean plasma concentration of 2-30 μM. In some embodiments, cisplatinis administered at a dosage sufficient to achieve a peak plasmaconcentration of 30-50 μM.

In embodiments where both auranofin and cisplatin are used, the dosageof auranofin is as described above, and the dosage of cisplatin is asdescribed above.

In some embodiments, the dosage of BSO and the metallodrug can bedefined as a ratio, wherein the amount of the metallodrug remains as aclinically-acceptable amount. In the present invention, the ratio of BSOto metallodrug is decreased by reducing the amount of BSO 10-1000-fold.In some embodiments, the ratio of BSO to metallodrug is 1:0.25 to 1:25.In some embodiments, the ratio of BSO to metallodrug is 1:0.25 to 1:13,1:0.25 to 1:10, 1:0.25 to 1:5, 1:0.25 to 1:1, 1:0.25 to 1:0.5. In someembodiments, the ratio of BSO to metallodrug is 1:1 to 1:2, 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. In some preferred embodiments, themetallodrug is a gold-containing metallodrug and the ratio of BSO tometallodrug is 1:1 to 1:10. In such embodiments, the gold-containingmetallodrug may be auranofin. In other embodiments, the metallodrug maybe a gold-containing metallodrug and the ratio of BSO to metallodrug is1:0.25, 1:0.5 or 1:1. In such embodiments, the gold-containingmetallodrug may be auranofin. In some embodiments, the metallodrug is aplatinum-containing metallodrug and the ratio of BSO to metallodrug is1:5 to 1:25, or, more preferably, 1:13 to 1:25. In such embodiments, theplatinum-containing metallodrug may be cisplatin.

In some embodiments, two or more metallodrugs can be administered withBSO, and the ratio of each metallodrug to BSO may be as defined above.In some embodiments, the ratio of BSO to a first metallodrug to a secondmetallodrug is 1:1-13:5-25. In some embodiments, BSO is administeredwith auranofin and cisplatin, and the ratio of BSO to auranofin tocisplatin is 1:1-3:5-25.

BSO and a metallodrug are useful for treatment via an anti-inflammatoryeffect and/or a cytotoxic effect. In particular, the anti-inflammatoryeffect involves a reduction in immune hyperactivity. Ananti-inflammatory effect can be determined, for example, by measuringlymphocyte activation markers such as the percentage of CD38+HLADR+CD8+T-cells. A cytotoxic effect can be determined, for example, by measuringneoplastic mass. In the case of HIV, the cytotoxic effect can bedetermined, for example, by measuring the amount of HIV DNA inperipheral blood mononuclear cells.

BSO and the metallodrug can be used for the treatment of cancer,rheumatic diseases or HIV, amongst others. Either, or both, thecytotoxic effect and the anti-inflammatory effect can be used to treatcancer. Rheumatic diseases and HIV are treated via the anti-inflammatoryeffect. In some embodiments, the cancer is a hematological malignancy,Hodgkin's or non-Hodgkin lymphoma, lung carcinoma, prostate cancer,hepatocellular carcinoma, breast cancer, glioblastoma, uterine cervixcarcinoma, or Kaposi's sarcoma. The rheumatic disease may be rheumaticarthritis or lupus. HIV may be HIV-1.

As discussed above, the metallodrug is administered at aclinically-acceptable amount, and this also includes the selection of anappropriate metallodrug for treating the disease in question. Thus, insome embodiments, the disease to be treated is cancer and themetallodrug is a gold-containing metallodrug, a platinum-containingmetallodrug, a zinc-containing metallodrug and/or a silver-containingmetallodrug. Preferably, the metallodrug is a gold-containingmetallodrug and/or a platinum-containing metallodrug. In suchembodiments, the metallodrug may be cisplatin, carboplatin, oxaliplatin,nedaplatin, lobaplatin, heptaplatin satraplatin, auranofin,aurothioglucose, sodium aurothiopropanolsulfonate and/or aurothiomalate.In preferred embodiments, the metallodrug is cisplatin and/or auranofin.In other embodiments, the disease to be treated is a rheumatic diseaseor HIV, and the metallodrug is a gold-containing metallodrug.

In some embodiments, BSO is used, with a metallodrug, for treatment viathe cytotoxic effect. The disease to be treated via the cytotoxic effectmay be cancer. In some embodiments of treatment via the cytotoxiceffect, BSO is administered at a dosage of 0.45-9 mg/kg, and themetallodrug is administered at a clinically-acceptable amount. Thedosage of the BSO may be such as to achieve a mean plasma concentrationof 0.05-0.8 μM when administered to the patient. In some embodiments,the metallodrug is auranofin. In such embodiments, the ratio of BSO toauranofin may be 1:1 to 1:10. In other embodiments, the metallodrug iscisplatin. In these embodiments, the ratio of BSO to cisplatin may be1:5 to 1:25. In other embodiments, the ratio of BSO to cisplatin is 1:13to 1:25. In some embodiments, the metallodrug is auranofin andcisplatin, and the ratio of BSO to auranofin to cisplatin is1:1-10:5-25.

In some embodiments, BSO is used, with a metallodrug, for treatment viathe anti-inflammatory effect. In some such embodiments, BSO isadministered at a dosage of 0.45-45 mg/kg, and the metallodrug isadministered at a clinically-acceptable amount. The dosage of the BSOmay be such as to achieve a mean plasma concentration of 0.05-2 μM. Insome embodiments, the metallodrug is auranofin. In some embodiments, theratio of BSO to auranofin is 1:0.25 to 1:1, preferably 1:0.25. In someembodiments, cancer, a rheumatic disease or HIV, such as those describedabove, is treated via the anti-inflammatory effect.

BSO can be used, with a metallodrug, together with existing therapiesfor the disease in question. Thus, in some embodiments, BSO and ametallodrug can be used together with an optimised background therapy,which may be an optimised background therapy for cancer, HIV or arheumatic disease. In some embodiments, BSO is administered in cycles,intertwined with periods of treatment with optimised background therapyand/or a metallodrug. The use of BSO may comprise a structured treatmentinterruption (STI) involving the interruption of administration of alltreatments with the optimised background therapy and/or the metallodrug.In some embodiments, the BSO is administered in cycles and the STI is atthe end of the final cycle of BSO administration.

In embodiments where the BSO and metallodrug are used to treat HIV, thepatient may be receiving antiretroviral therapy (ART).

The invention also provides a method of treating a patient in needthereof with BSO and a metallodrug. The BSO and the metallodrug may beadministered separately, simultaneously or sequentially. The BSO isadministered at a dosage less than or equal to 50 mg/kg, or any otherdosage described above. The metallodrug is administered at aclinically-acceptable dosage, as described above. The BSO and themetallodrug may be administered at a dosage ratio described above. Themethod of treatment may be for the treatment of a patient via thecytotoxic effect of BSO with a metallodrug, or via the anti-inflammatoryeffect of BSO with a metallodrug, as described above. In particular, themethod of treatment may be for the treatment of cancer, a rheumaticdisease or HIV.

The invention also provides a kit comprising BSO and a metallodrug. Thekit is for administering BSO and a metallodrug as described above. Thus,the kit comprises BSO at a dosage of less than or equal to 5000 mg, anda metallodrug at a clinically-acceptable dosage. The dosages of the BSOand the metallodrug in the kit are such that the ratio of BSO tometallodrug in the kit is a ratio disclosed above, when the metallodrugis at a clinically-acceptable dosage. The kit allows for theadministration of BSO at a dosage of less than or equal to 50 mg/kg, orany other dosage as described above. The dosage of the metallodrug, inthe kit, may also in accordance with the dosages described above. Thekit is useful for the treatment of cancer, rheumatic diseases and HIV,as described above.

The invention also provides for a composition comprising both BSO and ametallodrug, and a pharmaceutically-acceptable carrier, diluent and/orexcipient. Such pharmaceutically-acceptable carriers, diluents andexcipients are known to those skilled in the art. The compositionprovides for simultaneous administration of both BSO and themetallodrug, in a single dosage form. In some embodiments, the BSO andthe metallodrug are present in a single solution. In some embodiments,the BSO and metallodrug are present in a solid form. The solid form maybe reconstituted before administration to the patient. In someembodiments, the composition is administered by continuous infusion.

The amounts of BSO and the metallodrug in the composition are asdescribed above. Thus, the composition comprises BSO at a dosage of lessthan or equal to 50 mg/kg, or any other dosage described above, and themetallodrug is present at a clinically-acceptable dosage, as describedabove. For example, in some embodiments, the composition may comprise nomore than 9 mg/day, preferably 3-9 mg of a metallodrug, such asauranofin. Alternatively, the composition may comprise no more than 100mg/m², preferably between 30-100 mg/m² of a metallodrug, such ascisplatin. The ratio of BSO to the metallodrug is as described above.For example, the ratio of BSO to metallodrug may be 1:0.25 to 1:25, whenthe metallodrug is at a clinically-acceptable dosage. In someembodiments, the metallodrug is auranofin and the ratio of BSO toauranofin is 1:0.25 to 1:10. In other embodiments, the metallodrug iscisplatin and the ratio of BSO to metallodrug is 1:13 to 1:25.

The composition can comprise more than one metallodrug, and the morethan one metallodrug can be as described above. The amounts and ratiosof BSO and the more than one metallodrug are also as described above.

The composition is for the treatment of diseases as disclosed above.Cancer, rheumatic diseases and HIV are particularly preferredindications, and any of the diseases described above can be treated withthe compositions of the invention.

BSO and the metallodrug can be administered using any suitable deliverytechnique known to those skilled in the art. For example, among othertechniques, BSO and the metallodrug can be administered to a patientorally, by intra-venous infusion or by injection, such as intramuscularinjection.

In embodiments where BSO and a metallodrug are provided in a singlecomposition, the composition is preferably administered by intramuscularinjection.

EXAMPLES Example 1

In-Vitro Activity on Activated CD4⁺ T-Cells and Leukemia/Lymphoma

Methods. Resting CD4⁺ T-cells, cultured T-cell leukemic Jurkat cells andSup-T1 cells were left untreated or incubated for 24-72 hours withvarious concentrations of auranofin and/or buthionine sulfoximine (BSO),or chloroquine (only CD4⁺ T-cells) or cisplatin (only Jurkat and Supt-T1cells) CD4⁺ T-cells were then activated by adding phytohemagglutinin (2μg/mL). Alternatively, CD4⁺ T-cells were first activated withanti-CD3/anti-CD28 beads and, after 72 hours, were left untreated orwere incubated with auranofin and/or buthionine sulfoximine (BSO), orchloroquine. Cell viability was determined 24-72 hours post-activationwith an MTT assay (CellTiter 96® Non-Radioactive Cell ProliferationAssay) or MTS assay (CellTiter 96® AQueous One Solution CellProliferation Assay System; Promega). For the assay, 300×10⁵ cells werere-suspended in 100 μL of medium and transferred to a 96-well plate.Cells were then incubated with 20 μL of a substrate solution (OneSolution Reagent) for 3-4 hours in a CO₂ (5%) incubator at 37° C. Forthe MTT assay, 100 μL of a Solubilization/Stop Solution were used tostop the reaction. Medium-only containing wells were included to serveas a blank control. Absorbance levels were recorded at 490 nm (MTS) or570 nm (MTT) and, after subtraction of the blank, absorbance values wereexpressed as percentage of controls.

Results. The results from the new dosage combinations were compared tothe untreated cells as well as the cells treated with single drugs or“classical” dosages, in a multiple comparison analysis. Thus, thestatistical significances shown are also significant compared to theuntreated cells.

The results in FIG. 1 clearly show that concentrations as low as 2 μM ofBSO have a synergistic effect with auranofin in limiting the viabilityof activated CD4⁺ T-cells in a manner similar to a classically-describedconcentration (250 μM), and with an effect superior to that observedusing clinically achievable concentrations of Chloroquine. Chloroquineis an immune-modulating drug being investigated against malignant immunehyperactivation in HIV/AIDS (Savarino and Shytaj, 2015). These effectswere visible both when cells were pre-incubated with the drugcombination (FIG. 1A) and when they were treated concomitantly toactivation (FIG. 1B). FIG. 1B shows that the effects of BSO are stillpresent when the drug is used at submicromolar concentrations.

The results displayed in FIGS. 2 and 3 show that submicromolarconcentrations of BSO induced a direct anticancer effect in thelymphoblastic Supt-1 cell line when used in combination withclinically-achievable auranofin concentrations. Surprisingly, asynergism with auranofin (synergistic index<0.9) was apparent only usingthe submicromolar range of BSO concentrations. In particular, althoughauranofin and BSO at a “classic” concentration of 250 μM showed lowviability, the effects of the drug combination were not clear becausethe cytotoxic effect seen were ascribable to the effects of 250 μM ofBSO only. Indeed, there was no significant difference between 250 μM ofBSO only and the same BSO concentration in the presence of auranofin.Instead, using submicromolar BSO concentrations, there was an extremelysignificant difference between the effects of the combined drugtreatments and the corresponding concentrations of the single drugsalone, yielding a synergistic index<0.9 (see, for example, BSO 0.75alone and auranofin 0.5 alone, compared to auranofin 0.5+BSO 0.75).

Although FIG. 2 shows a significant synergism in terms of cell viabilityonly with 0.5 μM of auranofin, FIG. 3 shows that 0.25 μM of auranofin incombination with submicromolar concentrations of BSO inducesmorphological alterations linked to poor lymphoblastic cell viability,such as disruption of clustering.

Significant synergism was also found with the combination of BSO andcisplatin, a metallodrug used in several chemotherapeutic protocols forleukemia/lymphoma treatment, in Jurkat T cells (a leukemic cell line;FIG. 4). In addition, the combination of auranofin and lowconcentrations of BSO was able to increase the anti-neoplastic effect ofcisplatin (FIGS. 4 and 5). The effects observed in terms of cellviability were ascribable to the chemosensitizing effect of BSO onsusceptibility to cisplatin (FIG. 4), and microscopic analysis of thetreated cells showed that further addition of auranofin at aclinically-acceptable concentration increased the aforementionedmorphological alterations linked to lymphoblastic cell death (FIG. 5).

A chemosentitizing effect of BSO on susceptibility to cisplatin was alsodetected in the Supt-1 cell line (FIG. 6). Surprisingly, in both celllines, the submicromolar concentrations of BSO in combination with aclinically-acceptable concentration of cisplatin (10 μM) induced aninhibition of cellular proliferation similar to that obtained with a“classic” supramicromolar BSO concentration (250 μM; no statisticaldifference between treatments). Although 250 μM of BSO increasedcisplatin-induced cell death, the effect was antagonistic (synergisticindex>1.1; FIG. 6). Instead, a true synergism was observed by combiningBSO at submicromolar concentrations and cisplatin at an average plasmaconcentration of 10 μM (FIGS. 4-6).

Example 2

Clinical Use

The following is a theoretical example for a typical situation whereinthe BSO is used at a reduced concentration, with a clinically-acceptabledosage of auranofin to treat a patient with an HIV diagnosis.

Patient XX (male, 28 y.o.) received an HIV diagnosis in 2008. His viralload and CD4⁺ T-cell counts resulted to be 48,000 plasma viral RNAcopies/mL and 430 cells/μL, respectively. A standard therapy consistingof tenofovir/emtricitabine and efavirenz is started. Three months later,the CD4⁺ T-cell count has risen to 680 cells/μL and viral load hasdropped to undetectable levels. The situation is maintained essentiallystable during the following nine years, and the only significant eventsis a switch from efavirenz to raltegravir and occasional viral blipswith viral loads never rising above 500 viral RNA copies/mL.

The patient is subjected to quantification of viral DNA, the result ofwhich is 22 copies/10⁶ CD4⁺ T-cells. The patient now starts a drugregimen comprising auranofin (6 mg bid for six months) and low-dose BSO(three one-week cycles at 0.08 g orally every 6 hours, intertwined by 3weeks off BSO starting at month 3 of auranofin treatment). Backgroundantiretroviral therapy is maintained during the entire six-month period.The computational simulations presented in Example 3 show that patientXX's viral reservoir decreases below the limit of detection of the mostsensitive techniques, i.e. 10 copies of viral DNA/million cells, thusrendering him eligible for a structured treatment interruption aimed atexploring whether he will be able to maintain viral load under checkspontaneously without any further therapeutic interventions.

Example 3

Computational Simulations.

Computational simulations of the latently HIV-infected cell dynamicswere based on the system of differential equations developed by Rong andPerelson (Rong and Perelson; 2009), applying the baseline parameters asshown by the same authors. Numerical simulations were performed with theordinary differential equations solver of the Mathlab software. Theproliferation-upon-activation rate (henceforth, proliferation rate) wasdecreased from 1.4 day⁻¹ (normal rate) to 0.56, 0.28 and 0.14 day⁻¹.

The results showed that the viral reservoir decayed when the lymphocyteproliferation rate was decreased, but, unexpectedly, its decay rate waspartially independent of the size of the decrease of the proliferationrate (FIG. 7). These results further support a scenario wherein low BSOdosages may be added to clinically-achievable auranofin dosages, andsuggest a possibility of therapeutic success also in case thispharmacological combination should not reach the same level of in vivoanti-lymphoproliferative efficacy caused by the “classical”auranofin/BSO combinations containing higher amounts of BSO.

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The invention claimed is:
 1. A method of treating malignant immunehyperactivation in a human, wherein the method comprises administering:(a) buthionine sulfoximine (BSO) at a dosage of less than or equal to 9mg/kg, and (b) a gold-containing metallodrug at a clinically-acceptabledosage.
 2. A method according to claim 1, wherein the malignant immunehyperactivation is cancer, HIV or a rheumatic disease.
 3. A methodaccording to claim 2, wherein the cancer is haematological malignancy,Hodgkin's lymphoma, non-Hodgkin's lymphoma, lung carcinoma, prostatecancer, hepatocellular carcinoma, breast cancer, glioblastoma, uterinecervix carcinoma or Kaposi's sarcoma.
 4. A method according to claim 1,wherein the BSO is administered at a dosage of between 0.45 mg/kg and 9mg/kg.
 5. A method according to claim 1, wherein the malignant immunehyperactivation is HIV or a rheumatic disease.
 6. A method according toclaim 1, wherein the malignant immune hyperactivation is rheumaticarthritis or lupus.
 7. A method according to claim 1, wherein thegold-containing metallodrug comprises auranofin, aurothioglucose and/oraurothiomalate.
 8. A method according to claim 1, wherein the in vivomolar ratio of BSO to gold-containing metallodrug is 1:0.25 to 1:10. 9.A method according to claim 8, wherein the malignant immunehyperactivation is cancer and the in vivo molar ratio of BSO togold-containing metallodrug is 1:0.5 to 1:10.
 10. A method according toclaim 8, wherein the malignant immune hyperactivation is HIV or arheumatic disease, and the in vivo molar ratio of BSO to gold-containingmetallodrug is 1:0.25 to 1:1.
 11. A method according to claim 1, whereinthe gold-containing metallodrug comprises auranofin administered at adosage of 3-9 mg/day.
 12. A method according to claim 1, wherein themalignant immune hyperactivation is HIV or a rheumatic disease, andwherein the method comprises administering to a patient in need thereof:(a) BSO at a dosage sufficient to achieve a peak plasma concentration of0.5-20 μM, and (b) a gold-containing metallodrug at aclinically-acceptable dosage.
 13. A method according to claim 1, whereinthe malignant immune hyperactivation is cancer, and wherein the methodcomprises administering to a patient in need thereof: (a) BSO at adosage sufficient to achieve a mean plasma concentration of 0.05-0.8 μM,and (b) a gold-containing metallodrug at a clinically-acceptable dosage.14. A kit comprising BSO and a gold-containing metallodrug, wherein: (a)the BSO is at a dosage of less than or equal to 900 mg, and (b) thegold-containing metallodrug is at a clinically-acceptable dosage.
 15. Apharmaceutical composition comprising BSO and a gold-containingmetallodrug, wherein the gold-containing metallodrug is present at aclinically-acceptable amount for administration to humans and the molarratio of BSO to the gold-containing metallodrug is 1:0.003 or lower, andwherein the pharmaceutical composition comprises apharmaceutically-acceptable carrier, diluent and/or excipient.